Some
thoughts about the digital enhancement of photomicrographic imagery

By
Paul James (UK)

It is assumed that the
reader has imaging software with the basic tools to enhance tonality etc.
There is therefore no point in praising

the virtues of one software package
or another, as there are so many available, and each have qualities that
suit individual tastes.

This article therefore concerns itself
with the principles of enhancing photographic imagery so that it appears
as close

to the original image perceived through
the microscope.

The following notes are brief, but
will hopefully guide those who are new to image enhancement techniques

I think the art of
optimising microscopic imagery, using well understood manipulative techniques
for live viewing, has been a vital part of image enhancement for many decades.
Recently the PC's ability to manipulate photo imagery has been recognised
by many individuals, who realise that when conservatively used, the relevant
software can genuinely aid their desire to clarify images captured either
on film or by the CCD.

Image enhancement can be used
in all sorts of ways, but basically there are a number of forms of manipulation
which are of importance in microscopic imagery :-

This is perhaps the most important
single facet of imaging manipulation, and where an image can be improved
considerably with a little understanding of tonal scales, and the relevant
use of software.

Brief
thoughts on tonality

In purely tonal terms, i.e..
without considering colour, an image can be made up from a range of greys
between white and black. In reality of course the intensity of tone between
two values is limitless, but because of technical problems, digital imaging
at present limits this range to a commonly accepted range of about 255
equally diminishing shades from white to black. It is best to think of
colour imagery in terms of just greyscale , as this makes the understanding
of tonal correction much easier to deal with.

Here's a typical example
of an image straight from the digital camera, which has been reduced in
size for convenience in this article. You will notice also a graph displaying
the proportions of light intensity present in this image, which contains
basically two areas of tone. Firstly, the black field boundary and some
darker leg parts show up as the the small peak at extreme left ( S ), and
secondly, the field's lighter tone cast is recorded strongly between (
M ) and ( H ) near the righthand side. Notice that there is little evidence
of any information in the mid tone areas.

Subjectively, the image appears
a little on the dark side, so by dragging with the mouse in the first instance
the highlight or white boundary ( H ), so that it meets with the outer
edge of the lighter field tones, we will have raised the tonal value of
the field to a more realistic level :-

In any bright field
imagery, the field is always going to be the brightest component of the
image and therefore we are going to make this appear as such. As an aside,
notice how the total number of individual tones have now fallen to 210.
In practise this is not too noticeable. Though the image of the field looks
more like the original, some tonal alteration on the specimen itself is
needed, which is still too dark. Since the darkest parts of the leg are
tonally identical to the original image, we must raise the value of the
middle tones, and this can be done by dragging the ( M ) cursor to the
left :-

The degree to which
we move this medium tone cursor depends on the subjective assessment of
the user, as there are no absolute values involved. There is a strong temptation
to overdo things in the first attempts of image manipulation, and it takes
a little while to understand just what is actually going on when we move
these cursors about.

I've also raised the
contrast by 9 %, but left brightness alone, so we now have a fairly realistic
image, which is similar to the original view through the microscope, although
the most difficult part, that of maintaining an evenly lit field in this
example required some 'handiwork,' involving the need to mechanically 'sweep'
away any faint yellowness on the left edge of the field boundary. This
was accomplished quickly with the 'air brush'( Subject of another article
)

Some
individuals would rather that the field was cropped in the first instance,
and I've

shown
below the tonal scales of the resulting image :-

Notice the minimal
presence of darker tones, and progressive intensity of medium to light
tones. The background field brightness is shown at the extreme right of
the graphic display, which reveals the gap caused by the loss of full brightness
from the previous stage above. This basic tonal 'shaping' of the graphic
display is fairly common in most BF microscopic imagery, though it must
be emphasised that wide variations will occur ; subject to the variations
of specimen density and also the presence of a black field boundary of
course.

Phase
Contrast imagery

Those
images captured from phase contrast set-ups do require some thought when
enhancing, as their tonal values differ significantly to those of BF. The
principal difference is seen in the field itself, which appears as a darker
hue, and the bulk of the light output in this sort of image is devoid of
intense light tones as in BF.

Here's a typical raw
digital camera image of Actinophrys in phase contrast. Note the tonal values,
the limitation of highlights and the virtual absence of the dark tones.
The range of tones is restricted in fact, but there is a very small presence
of dark features in the axial filaments, the sum total of which is so small
that they are only apparent as an isolated thin black line hard against
the left side of the graphic display. Moving the (S) cursor towards the
left edge of the peak mid grey output on the graph as might be done for
a BF image results in :-

The result is not
surprising since we have put the peak output of light closer to the dark
tone extremes.

A more realistic image
is obtained by partially moving the cursor as shown, and yields a reasonably
faithful copy of the original imagery as seen through the 'scope. The mid
grey cursor (M) can safely be left.

Darkfield
imagery

This
requires more consideration and expertise at the image capturing stage
and therefore will be dealt with in a later article.

Summary

The manipulation
of tonal intensities is very important in any photographic image, and though
the examples I've shown above are somewhat limited, and do not cover the
full spectrum of either microscopic imagery, or that of the software variations
themselves, there is I hope sufficient information above to get a new comer
on the road to understanding the processes involved.

I hope
to cover the remainder of the post enhancement techniques in later articles.